Effects of FGIN-1-27 on Pigmentation in Zebrafish

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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Zebrafish have melanin pigments on the surface, which allows simple observation of pigmentation without complicated experimental procedures (Choi et al., 2007). PTU, a potent inhibitor of melanogenesis, is used widely in zebrafish research (Elsalini and Rohr, 2003). In the present study, PTU was used as a positive control. As shown in Figure 6, FGIN-1-27 significantly inhibited the body pigmentation of zebrafish, similar to PTU.

FGIN-1-27 Reduced UVB-Induced Hyperpigmentation in Guinea Pig Skin

UVB-induced hyperpigmentation model in brown guinea pigs was used to examine the whitening effect of FGIN-1-27 in vivo. As shown in Figure 7A, representative photographs of guinea pig skin indicated that FGIN-1-27 (1%) significantly suppressed pigmentation when compared with the vehicle treatment. To further assess the degree of pigmentation, we checked the L value (brightness index) using a Spectrophotometer. The ΔL value of the FGIN-1-27 group was markedly higher than that of the vehicle group after 3 weeks of treatment, suggesting that FGIN-1-27 reduced UVB-induced hyperpigmentation in guinea pig skin (Figure 7B). Masson–Fontana ammoniacal silver staining of skin tissue showed that FGIN-1-27 significantly inhibited UVB-induced pigmentation in the epidermal basal layer (Figure 7C). Immunohistochemical staining of a melanocyte marker protein, S-100, revealed that melanocyte count was not affected by FGIN-1-27 (Figures 7D, E). These results demonstrate that FGIN-1-27 has whitening effects on UV-induced hyperpigmentation in vivo.

DISCUSSION

According to Global Industry Analysts, the global whitening market will reach $31.2 billion by 2024 (Kim et al., 2019). Many research groups are focusing their efforts to elucidate novel and effective whitening compounds. Although plenty of agents have been developed, only a few were demonstrated to be therapeutically effective due to cytotoxicity and weak efficacy (Kim et al., 2008; Singh et al., 2016). Thus, it is necessary to continue to discover more efficient and safer skin-whitening agents.

In the current study, FGIN-1-27 inhibited basal melanogenesis and reversed α-MSH, OAG, or ET-1-induced melanin increase, without affecting cell viability (Figures 1 and 5). Tyrosinase, TRP-1, and TRP-2 are the key enzymes in melanogenesis, while α-MSH and ET-1 promote pigmentation by increasing the expression of these three crucial melanogenic enzymes (Rzepka et al., 2016; Corre et al., 2004; Regazzetti et al., 2015). Our results suggested that FGIN-1-27 suppressed α-MSH or ET-1-induced tyrosinase, TRP-1, and TRP-2 expression increase (Figures 2A, B). Tyrosinase activity is critical to melanogenesis (Rzepka et al., 2016). 1-Oleoyl-2-acetyl-sn-glycerol (OAG) is a synthetic, membrane-permeable diacylglycerol (DAG) analog demonstrated to increase the activity of tyrosinase (Thébault et al., 2005). Interestingly, FGIN-1-27 markedly inhibited OAG-induced tyrosinase activity increase, and the expression of tyrosinase did not significantly change after 12 h of treatment (Figures 2C, D). Mushroom tyrosinase activity assay showed that FGIN-1-27 did not directly inhibit tyrosinase activity, which suggested that FGIN-1-27 was not a direct inhibitor of tyrosinase (Figure 2E). Microphthalmia-associated transcription factor (MITF) is a master transcription factor for melanogenesis and upregulates the expression of tyrosinase, TRP-1, and TRP-2 (Levy and Fisher, 2011; Kawasaki et al., 2008). The present studies showed that FGIN-1-27 suppressed basal, α-MSH, and ET-1-induced MITF expression increase (Figure 3). As mentioned above, FGIN-1-27 inhibits melanogenesis by decreasing the expression of MITF, tyrosinase, TRP-1, and TRP-2 and inhibiting the tyrosinase activity, which is contradictory with previous studies that suggested MDR activation can increase melanogenesis (Lv et al., 2019). There are two possible explanations for this effect. Given the opposing functional activities of FGIN-1-27 vs. diazepam, we speculate that FGIN-1-27 is an MDR inverse agonist rather than generally considered as an agonist in melanocytes. Furthermore, many mechanisms are also involved in the effect of FGIN-1-27 at times, perhaps, obscuring the role of MDR activation. Further comprehensive studies are needed to disclose the function and underlying mechanism of the FGIN-1-27 and MDR in melanocytes. 

Excessive UV irradiation is considered to be an important cause of skin darkening (Abdel-Naser et al., 2003). After exposure to UV radiation, keratinocytes, and melanocytes were activated and produced α-melanocyte-stimulating hormone (α-MSH), diacylglycerol (DAG), and endothelin-1 (ET-1) (D’Mello et al., 2016; Bae-Harboe and Park, 2012). α-MSH, ET-1, and DAG affect melanin synthesis through intracellular signaling pathways. When α-melanocyte-stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R), the intracellular level of cAMP is elevated and the PKA/CREB pathway is activated, finally promoting melanogenesis (Corre et al., 2004; Rzepka et al., 2016). OAG could activate the PKC-β, which phosphorylates serine residues on the cytoplasmic domain of tyrosinase and activates it (Kim et al., 2006; Kawaguchi et al., 2012; Yuan and Jin, 2018). MAPK signaling pathways including extracellular p38, ERK, and JNK, could regulate melanin synthesis (Zhou et al., 2014). The activation of the p38 signaling pathway decreased the expression of MITF and promotes melanogenesis (Hirata et al., 2007). The role of ERK and JNK pathway in melanogenesis remains controversial (Lee et al., 2013; Peng et al., 2014). Endothelin-1 (ET-1) was reported to induce melanogenesis via the activation of ERK and p38 (Park et al., 2015; Regazzetti et al., 2015). Besides, the cross-talk between PKA and PKC-β could amplify the melanogenic effect and MAPK provides meeting points for cross-talk between these signaling pathways (Lee and Noh, 2013). A great deal of attention has continuously focused on the development of novel skin-whitening agents, which inhibited melanogenesis by regulating pigmentation-related signaling pathways. Kim et al. research indicated that piperlonguminine inhibited PKA/CREB-mediated melanogenesis but did not affect PKC-mediated melanogenesis (Kim et al., 2006). Furthermore, having A decreased melanogenesis via affecting ERK pathways, but had no effects on PKA/CREB pathways (Fujimoto et al., 1988). In the present study, FGIN-1-27 decreased the expression of PKC-β, p-PKA cat, p-CREB, p-p38, and p-ERK (Figures 4 and 5). These results suggested that all three of the above-mentioned signaling pathways involving melanogenesis were inhibited after FGIN-1-27 was treated. This can explain why FGIN-1-27 inhibited α-MSH, ET-1, or OAG-induced melanogenesis.

Furthermore, we investigated the effects of FGIN-1-27 on the melanogenesis of zebrafish. Zebrafish is a highly beneficial vertebrate model organism because its organ system and gene sequence are similar to those of humans (Choi et al., 2007). Furthermore, zebrafish have melanin pigments on the surface, which allows simple observation of pigmentation without complicated experimental procedures (Kim et al., 2008). In the present study, FGIN-1-27 significantly decreased the body pigmentation in zebrafish (Figure 6), which is contradictory with previous studies that suggested MDR activation slightly increased the number of melanocytes in larval zebrafish (Allen et al., 2020). The possible reason is that there exists a different mechanism in the anti-melanogenic effect of FGIN-1-27. Further comprehensive research is needed to elicit the role of FGIN-1-27 in melanogenesis and melanocyte production in zebrafish. We also investigated the effects of FGIN-1-27 on melanogenesis in the skin of guinea pigs. As shown in Figure 7, we found that the topical application of FGIN-1-27 to the dorsal skin of a guinea pig in which hyperpigmentation had been induced by exposure to UVB, resulted in efficient whitening effects. These results suggested that FGIN-1-27 inhibited melanin production in active melanocytes, but not a reduction of the number of melanocytes.

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REFERENCES

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